Abstract
Purpose :
A cellular hallmark of inherited retinal degenerative diseases is progressive loss of photoreceptors until one is completely blind. Unlike mammalian models, Zebrafish (Zf) have the capacity to regenerate neurons following retinal insult. The goal of this study is to understand the mechanisms of rod photoreceptor regeneration in Zf and to identify the master regulatory genes crucial for this process. We hypothesize that single cell (SC) analysis of a Zf model replicating rod degeneration pathology of Retinitis Pigmentosa will allow us to assess the trajectory of cells responsible for rod regeneration.
Methods :
SC RNA sequencing analysis was performed on WT and P23H rhodopsin transgenic Zf retinas. UMAP and trajectory analyses were performed in Seurat and Monocle 3. DrivAER analysis was performed to identify master regulators involved at each stage of regeneration. Morpholinos and siRNAs were used to knock down candidate genes by intravitreal injection in adult retina. BrdU labeling was used to trace newly-formed cells. Immunohistochemistry was performed to assess impact on regeneration.
Results :
UMAP clustering revealed all cell types in both WT and P23H data, including a group of cells identified as retinal progenitor cells (RPCs). In the P23H dataset, these RPCs were part of a unique trajectory predicted to support rod regeneration. BrdU pulse-chase experiments show RPCs in the ONL differentiate into rods and have a turnover rate within 1 week. DrivAER analysis revealed master regulator genes at each stage of regeneration: proliferation, differentiation, and maturation of RPCs into rods. Knockdown of the predicted master regulator prdm1a reduced the number of cells expressing rhodopsin; however, RPC proliferation is still present. Furthermore, the remaining rods in these injected retinas showed more frequent neurite outgrowth as compared to the control.
Conclusions :
The unique trajectory predicted in the P23H model for rod regeneration may indicate that RPC’s present in WT retinas are in a quiescent state. In the P23H model, where constant degeneration and regeneration are occurring, these RPCs may be continuously activated and the predominant source for replenishing lost rods. Pulse-chase experiments indicate that degeneration and regeneration are rapid in our model. Knockdown experiments have helped validate our analysis that prdm1a plays an important role in rod gene expression.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.